Methods

BMDM from wild type (WT), MKK3−/−, and MKK6−/− mice were pre-treated with p38 inhibitor
SB203580 (SB), JNK inhibitor SP600125 (SP), and/or ERK inhibitor PD98059 (PD) and
stimulated with LPS. Supernatant protein levels were measured by multiplex bead immunoassay.
mRNA expression was determined by qPCR and protein expression by Western blot analysis.
De novo IL-10 mRNA synthesis was quantified in cells treated with ethynyl-uridine
and LPS followed by reverse transcription and qPCR. mRNA half-life was measured in
LPS-treated cells that were then incubated with actinomycin D ± SB203580.

Results

Pre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 production
in the three groups, while ERK and JNK inhibitors had minimal effects. IL-10 production
was significantly decreased in MKK3−/− BMDM compared with either WT or MKK6−/− cells.
IL-10 mRNA expression was modestly reduced in MKK3−/− BMDM but was preserved in MKK6−/−
cells compared with WT. De novo IL-10 mRNA synthesis was inhibited in MKK3−/− and
p38 inhibitor pre-treated cells, but not MKK6−/− cells compared with WT. IL-10 mRNA
half-life was markedly reduced in p38 inhibitor-treated WT cells while MKK-deficiency
had minimal effect. DUSP1 mRNA levels were preserved in MKK-deficient cells but not
in p38 inhibitor-treated WT cells. Tristetraprolin mRNA and protein levels were reduced
in p38 inhibitor-treated WT cells compared with MKK6−/− cells.

Conclusion

Unlike p38-inhibition, the absence of MKK6 mostly preserves IL-10 and TTP protein
expression in BMDM. MKK6-deficiency also spares DUSP1 and IL-1RA, which are key negative
regulators of the inflammatory response. Together, these data suggest that MKK6 is
a potential therapeutic target in RA.